Voltage-gated sodium (Na) channels (Nav) are complex integral membrane proteins that open by depolarization, allowing the influx of Na+ions which, in turn, mediate the fast depolarization phase of an action potential in many excitable cells, e.g., neurons, neuroendocrine cells, and cardiac and skeletal myocytes. The nine known alpha pore-forming Nav subunits that have been functionally expressed are classified into two major pharmacological groups: Nav that are either i) sensitive or ii) insensitive to tetrodotoxin (TTX), a lethal toxin isolated from puffer fish (Fugu. sp). TTX-sensitive (TTX-S) channels are blocked by low nM concentrations of TTX while TTX-resistant (TTX-R) channels are blocked by μM concentrations of TTX. Members of the TTX-S class include SCN1a (Nav1.1), SCN2a (Nav 1.2), SCN3a (Nav1.3), SCN4a (Nav1.4), SCN8a (Nav1.6), and SCN9a (Nav1.7). Members of the TTX-R class include SCN5a (Nav1.5), SCN10a (Nav1.8), SCN12a (Nav1.9) (Clare et al. (2000); Goldin et al. (2000)). SCN6a/SCN7a has not been functionally expressed; however, it is predicted to be TTX-sensitive since it contains an aromatic amino acid (Y) in the pore region of domain I known to be required for TTX blockade (Akopian et al. (1997)). Nav alpha subunits are very large and share features with calcium channels and the prototype Kv potassium channels first described in Drosophila (Fozzard and Hanck (1996)). It is believed that channels in this large super-family are formed by the association of four similar domains, each with six putative transmembrane segments (S1–6) and a pore (P) domain. In the case of the classical Na and Ca channels, these four domains are combined in a single gene: Domains I–IV (Plummer and Meisler (1999)). Nav alpha subunits form complexes with one or two beta subunits, probably through covalent interactions (Isom (2000); Isom (2001)). A variety of toxins have been shown to bind to other sites on Nav channels, including site-2 toxins that bind to site-2 and lead to persistent activation (e.g. veratridine and batrachotoxin). Local anesthetics interact with amino acids in the S6 transmembrane region of domain IV, which, by analogy to the crystallized K channel KcsA (Doyle et al. (1998)), are thought to line the pore (Clare et al. (2000)).
From a therapeutic perspective, pharmacological and kinetic differences of Nav isoforms provide a basis for developing tissue-specific therapeutic agents. For example, some local anesthetic agents (e.g. lidocaine) have a greater efficacy in the heart than in the nervous system, while guanidinium and μ-CTX toxins discriminate between heart, skeletal and nerve Na channels (Fozzard and Hanck, (1996)). Other antagonists have been found to block Na channel activity in a use-dependent manner by binding to specific channel conformations presented in closed, activated or inactivated states. These use-dependent blockers target aberrantly hyperactive channels in certain human diseases and thus, can be utilized to assist rational therapeutic development. However, these are rare examples of Nav subtype-specific antagonists. Fortunately, molecular identification and pharmacological characterization of channels underlying endogenous Na currents in cells may enable the association of specific Nav subtypes to specific diseases. Aberrant Nav expression has been identified as a contributing factor to human disease and debilitation including epilepsy, long QT syndrome, and paralysis. Recent investigation has implicated aberrant Nav expression as contributing to neuropathic pain (reviewed by Clare et al., 2000). For example, examination of injured DRG neurons reveals enhanced expression of certain Nav channels including the TTX-sensitive Nav alpha subunit SCN3a. Following nerve injury, neurons of the Dorsal Root Ganglion (DRG) become spontaneously active, activate at lower thermal and mechanical stimuli intensities and fire repetitively to supra-threshold stimuli (Gurtu and Smith (1988)). The elevated spontaneous activity in injured DRG can be blocked by local anesthetics (Chabal et al. (1989); Tanelian and MacIver (1991); Devor et al. (1992); Sotgiu et al. (1992); and Matzner and Devor (1994)), a class of compounds known to target Nav, as well as TTX (Amir et al. (1999)). In addition, it has been observed that peripheral axotomy of sensory neurons leads to an increase in a TTX-S sodium current with a SCN3a-like kinetics, having a significantly faster recovery from inactivation (τ˜15 msec) compared to TTX-S sodium currents in control rat neurons (τ˜60 msec) (Cummins and Waxman (1997)). Noteworthy, in some skeletal muscle Nav channelopathies, including paramyotonia congenita, an increased rate of Nav recovery from inactivation appears to contribute to the hyperexcitability of skeletal muscle by reducing the refractory period (Chahine et al. (1996)).
Delayed hyperexcitability that develops following peripheral nerve injury (thought to underlie some types of “neuropathic pain”) correlates with novel Nav expression including up-regulation of the TTX-sensitive alpha subunit Nav1.3 (SCN3a) in both unmyelinated and myelinated sensory neurons (Waxman (1999)). Numerous studies have demonstrated that peripheral nerve injury increases Nav1.3 expression in rat DRG neurons (Waxman et al. (1994); Black et al. (1999); Dib-Hajj et al. (1999)). For example, intrathecal application of GDNF reversed the upregulation of Nav1.3 after spinal nerve ligation (method: Kim and Chung, 1992) and attenuated aberrant ectopic activity and neuropathic pain behavior (Boucher et al. (2000)). Relatedly, in cultured dissociated small nociceptive DRG neurons, addition of Nerve Growth Factor (NGF) results in down-regulation of SCN3a mRNA (Black et al. (1997)). SCN3a is believed to contribute to neuronal hyperexcitability as a result of its ability to rapidly “reprime” (recovery from inactivation) during the re-polarization phase of the action potential. For example, in small rat DRG neurons, increased expression of Nav1.3 after peripheral axotomy correlated with a switch from a TTX-S current with slow recovery from inactivation to a TTX-S current with a four-fold more rapid recovery (rapid re-priming), resulting in increased frequency of repetitive firing (Cummins and Waxman (1997)). Physiological properties (e.g. development of and recovery from inactivation) of the up-regulated Na channel in axotomized DRG are nearly identical to SCN3a when compared to SCN3a transiently expressed in certain cell types (Cummins et al. (2001)). Black and colleagues showed increased SCN3a immunoreactivity in adult rat small DRG neurons after axotomy of peripheral sciatic nerve processes but not dorsal rhizotomy (Black et al. (1999)). Furthermore, expression of a rapidly-re-priming Na current was restricted to peripherally, not centrally, axotomized small DRG neurons (Black et al. (1999)). Similarly, Chaplan and colleagues demonstrated by quantitative PCR up-regulation of Nav1.3 mRNA in lumbar sensory spinal ganglia isolated from diabetic rats and rats treated with the chemotoxic agent vincristine (Chaplan, Calcutt and Higuera, Journal of Pain (2001) 2(2):S1:21). Aberrant SCN3a expression following peripheral nerve injury also occurs in humans. Coward and colleagues demonstrated SCN3a (Nav1.3) immunoreactivity in a subset of peripheral nerve fibers from patients that had experienced peripheral or central nerve injury. Consistent with data obtained in rat neuropathic pain models, no detectable increase in soma labeling was observed after central avulsion (axotomy) in humans (Coward et al. (2001)). Whether SCN3a is up-regulated in human DRG neurons after peripheral axotomy requires further investigation.
When compared, the kinetic and pharmacologic properties of human (Chen et al. (2000)) and rat (Cummins et al. (2001)) recombinant SCN3a channels are similar. For example, the recovery from inactivation time constant is ˜20 msec when membrane potential is held at −90 mV for both human and rat receptors (compare FIGS. 4 and 5 of Chen et al. (2000) with FIG. 4 of Cummins et al. (2001)). The voltage dependence of activation and inactivation are similar as well (midpoints of activation were −23 and −25 mV for human and rat, respectively; half steady state inactivation potentials were −69 and −65 mV, respectively). The similarity of rat and human SCN3a functional properties supports the hypothesis that increased expression of SCN3a in injured human DRG will likely contribute to enhanced firing frequencies similar to those observed in injured rat DRG neurons. The beta subunit(s) associated with the up-regulated SCN3a channel in injured DRG neurons are unknown. At least two Na channel beta subunits (β1 and β3) are known to be expressed in DRG neurons (Oh et al. (1995); Coward et al. (2001); Shah et al. (2001)), and it has been reported that in the CCI model of neuropathic pain there is 20% up-regulation of β3 in small diameter DRG neurons (Shah et al. (2001)). Co-expression of β1, β2, β1+β2, or β3 with Nav1.3 revealed that Nav1.3 voltage dependence of activation was shifted +7 mV in the presence of only β3. Furthermore, β3 shifted the voltage dependence of inactivation to the right by +7 mV, and β1+β2 (but neither alone) shifted it by +5 mV (Cummins et al. (2001)). To date, β2 has not been detected in cultured rat DRG neurons (Black et al. (1996)). Examined collectively, the aforementioned study data provide strong evidence that over-expression of Nav 1.3 in injured DRG neurons contributes to the genesis and maintenance of neuropathic pain in animals, including humans.
Interestingly, PN1 (also known as SCN9a, hNE (NeuroEndocrine channel) and Nav1.7) is another TTX-sensitive Nav alpha subunit preferentially-expressed in rat and human injured DRG neurons, trigeminal ganglion neurons and sympathetic neurons (Toledo Aral et al. (1997)). PN1 has been reported to be up-regulated in small diameter sensory neurons up to three months following CFA-induced inflammation of peripheral receptive fields [England et al., Peripheral Nerve Society Abstract (1999)]. In SNS null mice, a 50% up-regulation of PN1 mRNA was suggested to compensate for the hypoalgesia caused by the absence of SNS in carrageenan-induced inflammation [Akopain et al., Nat. Neurosci, (1999) 2:541]. Examination of injured human DRGs reveals that regulation of PN1 is similar to that of TTX-R channels (Coward et al. (2001)). Furthermore, RT-PCR data suggest a positive correlation between up-regulation of Nav1.7 (and Nav1.3) and the metastatic potential of prostate tumor cell lines (Diss et al. (2001)). Therefore, inhibitors of Nav1.7 and Nav1.3 may have therapeutic potential in curbing metastasis in certain cancers including prostate cancer.
Unfortunately, conventional therapy for treating neuropathic pain in humans due to ectopic (spontaneous) Nav activity, including administration of analgesics, anticonvulsants and anti-arrhythmics, has proven sporadically effective with demonstrable side-effects as a consequence of non-specific, low-potency interactions at Nav targets. This fact, coupled with a growing population of neuropathic pain sufferers, reveals the importance and immediate need for Nav subtype-specific antagonists. Historically, however, it has been the difficulty in constructing cell lines that stably express Nav subtypes that has slowed target-driven therapeutic design (Clare et al. (2000)).